Composition comprising a pulmonary surfactant and a pde2 inhibitor

ABSTRACT

The invention relates to the combined administration of a pulmonary surfactant and a PDE2 inhibitor for the treatment of a disease in which pulmonary surfactant malfunction and/or phosphodiesterase 2 (PDE2) activity is detrimental.

FIELD OF APPLICATION OF THE INVENTION

The invention relates to the combination of certain known activecompounds for therapeutic purposes. The compounds used in thecombination according to this invention are known pulmonary surfactantsand known active compounds from the phosphodiesterase 2 (PDE2) inhibitorclass. Their combined use in the sense according to this invention fortherapeutic purposes has not yet been described in prior art.

PRIOR ART

ARDS (Adult Respiratory Distress Syndrome) is a descriptive expressionwhich is applied to a large number of acute, diffuse infiltrativepulmonary lesions of differing etiology if they are associated with asevere gas exchange disorder (in particular arterial hypoxemia) [G. R.Bernard et al.: Report of the American-European consensus conference onARDS: definitions, mechanisms, relevant outcomes and clinical trialcoordination; Intensive Care Medicine, 1994, 20:225232]. The expressionARDS is also used for IRDS (Infant Respiratory Distress Syndrome)because of the numerous common clinical and pathological features. If,in the case of IRDS, the lung surfactant deficiency caused by prematurebirth is predominant, then in the case of ARDS a lung surfactantmalfunction is caused by the disease of the lung based on differingetiologies such as inhalation of toxins or irritants (e.g. chlorine gas,nitrogen oxides, smoke), direct or indirect trauma (e.g. multiplefractures or pulmonary contusion), systemic reactions to inflammationsoutside the lung (e.g. hemorrhagic pancreatitis, gram-negativesepticemia), transfusions of high blood volumes or alternatively aftercardiopulmonary bypass. In patients suffering from ARDS, lung surfactantfunction is impaired (=surfactant malfunction) so that the alveolarsurfactant layer does not prevent lung atelectasis and does not maintainphysiologic lung functions required for oxygenation.

In the healthy lung, pulmonary endothelium regulates the exchange offluid, solutes, macromolecules, and cells between vascular and tissuespaces. With inflammation abound in ARDS, the endothelial barrierbecomes more permissive for exchange leading to interstitial andalveolar edema formation. This process leads to a further impairment ofoxygenation.

Presently, the therapy of ARDS mainly consists in the earliest possibleapplication of different forms of ventilation (e.g. raising of theoxygen concentration of the respiratory air) up to extracorporealmembrane oxygenation. The specific use of various ventilation techniqueshas only led to a small lowering of mortality and including the risk ofdamaging the lungs by ventilation with pressure and high FiO₂ (Fractionof Inspired Oxygen; proportion of oxygen in the respiratory air). Inparticular, ARDS patients whose lungs have been damaged by ventilationneed even higher pressures and higher FiO₂ to obtain an adequateoxygenation of the blood.

Because surfactant function is impaired in ARDS, surfactant replacementtherapy is thought to improve lung function and oxygenation in ARDS. Ithas also proven suitable to treat IRDS by introducing pulmonarysurfactant preparations into the lungs of the children concerned.WO01076619 describes the use of a pulmonary surfactant preparation forthe prophylaxis or early treatment of acute pulmonary diseases such asARDS, IRDS or ALI (Acute Lung Injury). WO03033014, Spragg R G et al.[Spragg R G et al. (2003) American Journal Respiratory and Critical CareMedicine 167: 1562] and Eaton S et al. [Eaton S et al. (2002) ExpertOpinion on Investigational Drugs 11: 37] describe that pulmonarysurfactants, in particular rSP-C surfactants, are useful in thetreatment of ARDS.

Asthma patients, in particular in acute status asthmaticus, suffer fromobstructed airways due to bronchoconstriction, inflammation, mucushypersecretion, and edema formation. Due to extravasation of plasma andproteins into the alveolar lumen and due to released proteases, andmucus the surfactant function is disturbed leading to atelectasis andimpaired ventilation [Hohlfeld J M et al. Dysfunction of pulmonarysurfactant in asthmatics after segmental allergen challenge. Am J RespirCrit Care Med 1999; Fuchimukai T et al. Artificial pulmonary surfactantinhibited by proteins. J Appl Physiol 1987, 62:429-437: Seeger W et al.Surfactant inhibition by plasma proteins: differential sensitivity ofvarious surfactant preparations. Eur Respir J 1993, 6:971-977]. Fatalasthma attacks end up with insufficient oxygenation resulting partlyfrom edema formation and impaired ventilation due to a lack of activesurfactant.

There is first evidence on the value of surfactant treatment of patientswith Asthma in a pilot study the patients were treated with surfactantinhalation after an asthma attack. Respiratory functions and oxygenationwere markedly improved in all patients [Kurashima Ket al. A pilot studyof surfactant inhalation in the treatment of asthmatic attack. Arerugi.1991 February ;40(2):160-3].

WO 01058423 describes the use of pulmonary surfactant for theprophylaxis or treatment of chronic pulmonary diseases in mammals suchas chronic obstructive pulmonary disease (COPD), asthma, cysticfibrosis, pulmonary fibrosis, pulmonary degeneration, chronic bronchitisand pulmonary emphysema.

Recent data show that PDE2 is one of the major enzymes found in bovineand porcine endothelial cells [Ashikaga T et al. Altered expression ofcyclic nucleotide phosphodiesterase isozymes during culture of aorticendothelial cells Biochem Pharmacol. 1997 Nov. 15;54(10):1071-9; KishiYet al. Phosphodiesterases in vascular endothelial cells. Adv SecondMessenger Phosphoprotein Res. 1992; 25:201-13; Koga S et al. TNFmodulates endothelial properties by decreasing CAMP. Am J Physiol. 1995May; 268(5 Pt 1):C1104-13; Lugnier C, Schini V B. Characterization ofcyclic nucleotide phosphodiesterases from cultured bovine aorticendothelial cells. Biochem Pharmacol. 1990 Jan 1; 39(1):75-84; Souness JE et al. Pig aortic endothelial-cell cyclic nucleotidephosphodiesterases. Use of phosphodiesterase inhibitors to evaluatetheir roles in regulating cyclic nucleotide levels in intact cells.Biochem J. 1990 Feb 15;266(1):127-32]. Inhibition of PDE2 reducesmonolayer permeability of porcine pulmonary artery endothelial cells[Suftorp N et al. Role of nitric oxide and phosphodiesterase isoenzymeII for reduction of endothelial hyperpermeability. Am J Physiol. 1996March;270; (3 Pt 1):C778-85]. Finally, Suttorp N. et al. [Suttorp N. etal. (1996) Atemwegs und Lungenkrankheiten, DustriVerlag, Vol. 22:560-566] describes the use of PDE2 inhibitors to block pulmonaryvascular leakage.

In the European patent application EP 0771799, the international patentapplication WO98/40384 and in the U.S. Pat. No. 5,861,396 purin-6-onederivatives are described as PDE2 inhibitors suitable for the treatmentof cardiovascular disorders, disorders of the vascular system and of theurogenital system.

SUMMARY OF THE INVENTION

It is the object of the present invention to make available apharmaceutical composition suited for prevention or reduction of theonset of symptoms of a disease, or for treatment or reduction of theseverity of a disease in which pulmonary surfactant malfunction and/orphosphodiesterase 2 (PDE2) activity is detrimental.

Surprisingly, it has now been found that the combined use of a pulmonarysurfactant and a PDE2 inhibitor fulfills these conditions.

Thus, the invention relates to pharmaceutical compositions comprising apulmonary surfactant in combination with a PDE2 inhibitor and to methodsfor preventing or reducing the onset of symptoms of a disease in whichpulmonary surfactant malfunction and/or phosphodiesterase 2 (PDE2)activity is detrimental, and to methods for treating or reducing theseverity of a disease in which pulmonary surfactant malfunction and/orphosphodiesterase 2 (PDE2) activity is detrimental.

Accordingly, the invention relates in a first aspect to the combined useof a pulmonary surfactant and a PDE2 inhibitor for preventing orreducing the onset of symptoms of a disease, or treating or reducing theseverity of a disease in a patient in need thereof, in which diseasepulmonary surfactant malfunction and/or phosphodiesterase 2 (PDE2)activity is detrimental.

In another aspect of present invention, there is provided the use of acombination of a pulmonary surfactant and a PDE2 inhibitor for thepreparation of a medicament for preventing or reducing the onset ofsymptoms of a disease, or treating or reducing the severity of a diseasein a patient in need thereof, in which disease pulmonary surfactantmalfunction and/or phosphodiesterase 2 (PDE2) activity is detrimental.

In another aspect of present invention, there is provided a method forpreventing or reducing the onset of symptoms of a disease in whichpulmonary surfactant malfunction and/or phosphodiesterase 2 (PDE2)activity is detrimental, or treating or reducing the severity of adisease in which pulmonary surfactant malfunction and/orphosphodiesterase 2 (PDE2) activity is detrimental by administering to apatient in need thereof an effective amount of (1) a pulmonarysurfactant and (2) a PDE2 inhibitor.

In another aspect of present invention, there is provided a method forpreventing or reducing the onset of symptoms of a disease in whichpulmonary surfactant malfunction and/or phosphodiesterase 2 (PDE2)activity is detrimental, or treating or reducing the severity of adisease in which pulmonary surfactant malfunction and/orphosphodiesterase 2 (PDE2) activity is detrimental by simultaneouslyadministering to a patient in need thereof an effective amount of apulmonary surfactant and a PDE2 inhibitor.

In another aspect of present invention, there is provided a method forpreventing or reducing the onset of symptoms of a disease in whichpulmonary surfactant malfunction and/or phosphodiesterase 2 (PDE2)activity is detrimental, or treating or reducing the severity of adisease in which pulmonary surfactant malfunction and/orphosphodiesterase 2 (PDE2) activity is detrimental by administering insuccession, close in time or remote in time, in any order whatever to apatient in need thereof an effective amount of a pulmonary surfactantand a PDE2 inhibitor.

In another aspect of present invention, there is provided apharmaceutical composition suited for a method for preventing orreducing the onset of symptoms of a disease in which pulmonarysurfactant malfunction and/or phosphodiesterase 2 (PDE2) activity isdetrimental, or for treating or reducing the severity of a disease inwhich pulmonary surfactant malfunction and/or phosphodiesterase 2 (PDE2)activity is detrimental, which pharmaceutical composition comprises acombination of an effective amount of a pulmonary surfactant and aneffective amount of a PDE2 inhibitor.

In another aspect of present invention, there is provided apharmaceutical composition suited for a method for preventing orreducing the onset of symptoms of a disease in which pulmonarysurfactant malfunction and/or phosphodiesterase 2 (PDE2) activity isdetrimental, or for treating or reducing the severity of a disease inwhich pulmonary surfactant malfunction and/or phosphodiesterase 2 (PDE2)activity is detrimental, which pharmaceutical composition comprises as afixed combination an effective amount of a pulmonary surfactant and aneffective amount of a PDE2 inhibitor, and optionally a pharmaceuticallyacceptable carrier. In particular, such a fixed pharmaceuticalcomposition for intratracheal or intrabronchial instillation ispreferred.

In another aspect of present invention, there is provided apharmaceutical composition suited for a method for preventing orreducing the onset of symptoms of a disease in which pulmonarysurfactant malfunction and/or phosphodiesterase 2 (PDE2) activity isdetrimental, or for treating or reducing the severity of a disease inwhich pulmonary surfactant malfunction and/or phosphodiesterase 2 (PDE2)activity is detrimental, which pharmaceutical composition comprises as afree combination an effective amount of a pulmonary surfactant andoptionally a pharmaceutically acceptable carrier and an effective amountof a PDE2 inhibitor and optionally a pharmaceutically acceptablecarrier.

In another aspect of present invention there is provided the use of apharmaceutical composition comprising a combination of a pulmonarysurfactant and a PDE2 inhibitor for the treatment of ALI, ARDS, IRDS orAsthma bronchiale.

In another aspect of present invention there is provided the use of acombination of a pulmonary surfactant and a PDE2 inhibitor for thepreparation of a medicament for the treatment of ALI, ARDS, IRDS orAsthma bronchiale.

In another aspect of present invention there is provided a method forpreparing a pharmaceutical composition suited for preventing or reducingthe onset of symptoms of a disease in which pulmonary surfactantmalfunction and/or phosphodiesterase 2 (PDE2) activity is detrimental,or for treating or reducing the severity of a disease in which pulmonarysurfactant malfunction and/or phosphodiesterase 2 (PDE2) activity isdetrimental, which method comprises the step: mixing an effective amountof a pulmonary surfactant and a PDE2 inhibitor with a pharmaceuticallyacceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION

The combination therapy, which is the subject matter of presentinvention, comprises administering a pulmonary surfactant with a PDE2inhibitor to prevent the symptoms or the onset of a disease or to treata disease in which pulmonary surfactant malfunction and/orphosphodiesterase 2 (PDE2) activity is detrimental.

The invention thus relates to the combined use of a pulmonary surfactantand a PDE2 inhibitor in preventing the symptoms of, or treating adisease in which pulmonary surfactant malfunction and/orphosphodiesterase 2 (PDE2) activity is detrimental.

The “pulmonary surfactant” useful in this invention may be any compoundor pulmonary surfactant preparation that is known to have the samesurface-active properties as natural pulmonary surfactant; naturalpulmonary surfactant reduces, for example, the surface tension in thealveoli.

A simple and rapid in vitro test with which the surface activity ofpulmonary surfactant can be determined is, for example, the so-calledWilhelmy balance [Goerke, J. Biochim. Biophys. Acta, 344: 241-261(1974), King R. J. and Clements J. A., Am. J. Physicol. 223: 715-726(1972)]. This method gives information on the pulmonary surfactantquality, measured as the action of a pulmonary surfactant of achieving asurface tension of almost zero mN/m. Another measuring device fordetermining the surface activity of pulmonary surfactant is thepulsating bubble surfactometer [Possmayer F. et al., Prog. Resp. Res.,Ed. v. Wichert, Vol. 18: 112-120 (1984)]. The activity of a pulmonarysurfactant preparation can also be determined by means of in vivo tests,for example as described by Häfner et al. [D. Häfner et al.: Effects ofrSP-C surfactant on oxygenation and histology in a rat lung lavage modelof acute lung injury. Am. J. Respir. Crit. Care Med. 1998,158: 270-278].

A group of known pulmonary surfactant preparations and theirmodifications that may be usefully as pulmonary surfactant employed inthe present invention include pulmonary surfactant preparations havingthe function of natural pulmonary surfactant. Preferred pulmonarysurfactant preparations are those which, for example, have activity inthe tests described above. Particularly preferred pulmonary surfactantpreparations are those which exhibit increased activity in such a testin comparison with natural, in particular human, pulmonary surfactant.In this context, these can be compositions which only containphospholipids, but also compositions which, apart from thephospholipids, inter alia additionally contain pulmonary surfactantprotein.

Preferred phospholipids according to the invention aredipalmitoylphosphatidylcholine (DPPC),palmitoyloleylphosphatidylglycerol (POPG) and/or phosphatidylglycerol(PG). Particularly preferably, the phospholipids are mixtures of variousphospholipids, in particular mixtures of dipalmitoylphosphatidylcholine(DPPC) and palmitoyloleylphosphatidylglycerol (POPG), preferably in theratio from 7 to 3 to 3 to 7.

Commercial products which may be mentioned as pulmonary surfactantpreparations are

-   -   CUROSURF® (INN: PORACTANT ALFA) (Serono, Pharma GmbH,        Unterschleiβheim), a natural surfactant from homogenized porcine        lungs;    -   SURVANTA® (INN: BERACTANT) (Abbott GmbH, Wiesbaden), extract of        bovine lungs;    -   ALVEOFACT® (INN: BOVACTANT) (Boehringer Ingelheim), extract of        bovine lungs;    -   EXOSURF® (INN: COLFOSCERIL PALMITATE) (Glaxo SmithKline), a        synthetic phospholipid containing excipients;    -   SURFACTEN® (INN: SURFACTANT-TA) (Mitsubishi Pharma Corporation),        a pulmonary surfactant extracted from bovine lungs;    -   INFASURF® (INN: CALFACTANT) (Forest Pharmaceuticals), a        surfactant extracted from calf lungs;    -   ALEC® (INN: PUMACTANT) (Britannia Pharmaceuticals), an        artificial surfactant of DPPC and PG; and    -   BLES® (BLES Biochemical Inc.), a bovine lipid extract        surfactant.

Suitable pulmonary surfactant proteins are both the proteins obtainedfrom natural sources, such as pulmonary lavage or extraction fromamniotic fluid, and the proteins prepared by genetic engineering orchemical synthesis. According to the invention, in particular thepulmonary surfactant proteins designated by SP-B (Surfactant Protein-B)and SP-C (Surfactant Protein-C) and their modified derivatives are ofinterest. The amino acid sequences of these pulmonary surfactantproteins, their isolation or preparation by genetic engineering areknown (e.g. from WO 8603408, EP 0251449, WO 8904326, WO 8706943, WO8803170, WO 9100871, EP 0368823 and EP 0348967). Modified derivatives ofthe pulmonary surfactant proteins designated by SP-C, which differ fromhuman SP-C by the replacement of a few amino acids, are described, forexample, in WO 9118015 and WO 9532992. Particularly to be emphasized inthis connection are the recombinant SP-C (rSP-C) derivatives which aredisclosed in WO 9532992, in particular those which differ from humanSP-C in positions 4 and 5 by the substitution of cysteine byphenylalanine and in position 32 by the substitution of methionine byisoleucine [designated herein as rSP-C (FF/I) or LUSUPULTIDE (INN) orVENTICUTE®]. Modified derivatives of pulmonary surfactant proteins arealso understood as meaning those proteins which have a completelyoriginally designed amino acid sequence with respect to their pulmonarysurfactant properties, such as are described in EP 0593094 and WO9222315. Preferably, the polypeptide KL4 (INN: SINAPULTIDE, SURFAXIN®)may be mentioned in this connection. The name pulmonary surfactantprotein, according to the invention, also comprises mixtures ofdifferent pulmonary surfactant proteins. In EP 0100910, EP 0110498, EP0119056, EP 0145005 and EP 0286011 phospholipid compositions with andwithout pulmonary surfactant proteins are described which are likewisesuitable as components of the preparations.

As further constituents which can be present in pulmonary surfactantpreparations, fatty acids such as palmitic acid may be mentioned. Thepulmonary surfactant preparations can also contain electrolytes such ascalcium, magnesium and/or sodium salts (for example calcium chloride,sodium chloride and/or sodium hydrogencarbonate) in order to establishan advantageous viscosity. Preferred pulmonary surfactant preparationsaccording to the invention contain 80 to 95% by weight of phospholipids,0.5 to 3.0% by weight of pulmonary surfactant proteins, 3 to 15% byweight of fatty acid, preferably palmitic acid, and 0 to 3% by weight ofcalcium chloride.

The pulmonary surfactant preparations are prepared by processes knownper se and familiar to the person skilled in the art, for example asdescribed in WO 9532992. According to the invention, the pulmonarysurfactant preparations are preferably lyophilized and in particularspray-dried pulmonary surfactant preparations. Lyophilized preparationsare disclosed, for example, in WO 9735882, WO 9100871 and DE 3229179. WO9726863 describes a process for the preparation of powdered pulmonarysurfactant preparations by spray drying. According to the invention,preparations prepared in this way are preferred.

According to this invention, the term “PDE2 inhibitor” refers to aselective phosphodiesterase (PDE) inhibitor, which inhibitspreferentially the type 2 phosphodiesterase (PDE2) when compared toother known types of phosphodiesterase, e.g. type 1, 3, 4, 5, etc.(PDE1, PDE3, PDE4, PDE5, etc.). According to this invention, a PDEinhibitor preferentially inhibiting PDE2 refers to a compound having alower IC50 for PDE2 (i.e. the IC50 for PDE2 inhibition is about 10 timeslower than the IC50 for inhibition of other known types ofphosphodiesterase, e.g. type 1, 3, 4, 5, etc.) and therefore is morepotent to inhibit PDE2.

For activity determination of PDE2, the [³H]CAMP SPA assay (AmershamLife Science) may be used −10⁻⁶M cGMP being added to the reactionmixture to activate the enzyme. Other methods for activity testing ofPDE2 inhibitors are disclosed in WO 9840384 and U.S. Pat. No. 5,861,396.It is also possible to determine PDE2 activity by the method describedby Tenor H et al. [Tenor H et al. (2002) British J Pharmacol 135: 609].

A group of PDE2 inhibitors that may be usefully employed in presentinvention include the purin-6-one derivatives as revealed in EP 0771799,WO98/40384 and in U.S. Pat. No. 5,861,396.

Compounds which may be mentioned as preferred examples of PDE2inhibitors are

-   N-Benzyl-2-[5-fluoro-2-methyl-1(Z)-(3,4,5-trimethoxybenzylidene)inden-3-yl]acetamide,-   2-(3′-Aminobiphenyl-4-ylmethyl)-9-(1-methyl-4-phenylbutyl)hypoxanthine,-   N-Benzyl-2-[5-fluoro-2-methyl-1(Z)-(3,4,5-trimethoxybenzylidene)inden-3-yl]acetamide,-   2-(3′-Aminobipheny-4-ylmethyl)-9-(1-methyl-4-phenylbutyl)hypoxanthine,-   2-Benzyl-9-(1-methyl-4-phenylbutyl)hypoxanthine,-   2-(3,4-Dichlorobenzyl)-9-[1-(1-hydroxyethyl)-4-phenylbutyl]hypoxanthine,-   2-(4-Fluorobenzyl)-9-(1-methyl-4-phenylbutyl)hypoxanthine,-   9-(1-Methyl-4-phenylbutyl)-2-[4-(3-thienyl)benzyl]hypoxanthine,-   1-[5-[9-[1-(1-Hydroxyethyl)-4-phenylbutyl]hypoxanthin-2-ylmethyl]-2-methoxyphenylsulfonyl]piperidine-4-carboxylic    acid,-   2-(Biphenyl-4-yl)-9-(1-methyl-4-phenylbutyl)-6,9-dihydro-1H-purin-6-one,-   2-(4-Chlorophenyl)-9-[1-(1-hydroxyethyl)heptyl]-6,9-dihydro-1H-purin-6-one,-   2-Cyclohexyl-9-[1-(1-hydroxyethyl)-4-phenylbutyl]-6,9-dihydro-1H-purin-6-one,-   2-Cyclopropyl-9-(1-methyl-4-phenylbutyl)-6,9-dihydro-1H-purin-6-one,-   2-(1,3-Benzodioxol-5-yl)-9-(1-methyl-4-phenylbutyl)-6,9-dihydro-1H-purin-6-one,-   erythro-9-(2-hydroxy-3-nonyl)adenine,-   9-(6-Phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one,-   6-(3,4-Dimethoxy-benzyl)-1-[1-(1-hydroxy-ethyl)-4-phenyl-butyl]-3-methyl-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one,-   N-benzyl-2-(6-fluoro-2-methyl-3-pyridin-4-ylmethylene-3H-inden-1-yl)-acetamide,-   (1Z)-N-benzyl-2-[6-fluoro-2-methyl-3-(3,4,5-trimethoxybenzylidene)-3H-inden-1-yl]-acetamide,-   N-Benzyl-2-[5-fluoro-2-methyl-1-[(Z)-(pyridin-4-yl)methylene]-1H-inden-3-yl]acetamide    hydrochloride,-   4-[N-[4-[9-[N-Methyl-N-(3-phenylpropyl)amino]hypoxanthin-2-ylmethyl]phenyl]carbamoyl]piperidine-1-carboxylic    acid benzyl ester,-   4-[N-[4-[9-(N-hexyl-N-methylamino)hypoxanthin-2-ylmethyl]phenyl]carbamoyl]piperidine-1-carboxylic    acid benzyl ester,-   2-(3,4-Dimethoxybenzyl)-9-[N-methyl-N-(3-phenylpropyl)amino]hypoxanthine,-   9-[N-Methyl-N-(3-phenylpropyl)amino]-2-[4-(4-methylpiperazin-1-ylsulfonyl)benzyl]hypoxanthine,-   2-(3,4-Dimethoxybenzyl)-7-[1(R)-[1(R)-hydroxyethyl]-4-phenylbutyl]-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one,-   7-(1-Acetylpentyl)-5-methyl-2-(4-methylbenzyl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one,-   7-(1-Acetylhexyl)-5-methyl-2-(4-methylbenzyl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one,-   2-(3,4-Dimethoxybenzyl)-7-[1-(1-hydroxyethyl)-5-hexenyl]-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one,    and-   and the pharmaceutically acceptable salts of these compounds.

Particularly preferred examples of PDE2 inhibitors are

-   erythro-9-(2-hydroxy-3-nonyl)adenine,-   9-(6-Phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one,-   6-(3,4-Dimethoxy-benzyl)-1-[1-hydroxy-ethyl)-4-phenyl-butyl]-3-methyl-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one,-   N-benzyl-2-(6-fluoro-2-methyl-3-pyridin-4l-ylmethylene-3H-inden-1-yl)-acetamide,-   (1Z)-N-benzyl-2-[6-fluoro-2-methyl-3-(3,4,5-trimethoxybenzylidene)-3H-inden-1-yl]-acetamide,-   4-[N-[4-[9-[N-Methyl-N-(3-phenylpropyl)amino]hypoxanthin-2-ylmethyl]phenyl]carbamoyl]piperidine-1-carboxylic    acid benzyl ester,-   4-[N-[4-[9-(N-hexyl-N-methylamino)hypoxanthin-2-ylmethyl]phenyl]carbamoyl]piperidine-1-carboxylic    acid benzyl ester,-   2-(3,4-Dimethoxybenzyl)-9-[N-methyl-N-(3-phenylpropyl)amino]hypoxanthine,-   9-[N-Methyl-N-(3-phenylpropyl)amino]-2-[4-(4-methylpiperazin-1-ylsulfonyl)benzyl]hypoxanthine,-   2-(3,4-Dimethoxybenzyl)-7-[1(R)-[1(R)-hydroxyethyl]-4-phenylbutyl]-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one,-   7-(1-Acetylpentyl)-5-methyl-2-(4-methylbenzyl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one,-   7-(1-Acetylhexyl)-5-methyl-2-(4-methylbenzyl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one,-   2-(3,4-Dimethoxybenzyl)-7-[1-(1-hydroxyethyl)-5-hexenyl]-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one,    and-   and the pharmaceutically acceptable salts of these compounds.

Salts encompassed within the term “pharmaceutically acceptable salts”refer to non-toxic salts of the compounds which are generally preparedby reacting a free base with a suitable organic or inorganic acid or byreacting the acid with a suitable organic or inorganic base. Particularmention may be made of the pharmaceutically acceptable inorganic andorganic acids customarily used in pharmacy. Those suitable are inparticular water-soluble and water-insoluble acid addition salts withacids such as, for example, hydrochloric acid, hydrobromic acid,phosphoric acid, nitric acid, sulfuric acid, acetic acid, citric acid,D-gluconic acid, benzoic acid, 2-(4-hydroxybenzoyl)-benzoic acid,butyric acid, sulfosalicylic acid, maleic acid, lauric acid, malic acid,fumaric acid, succinic acid, oxalic acid, tartaric acid, embonic acid,stearic acid, toluenesulfonic acid, methanesulfonic acid or1-hydroxy-2-naphthoic acid, the acids being employed in saltpreparation—depending on whether it is a mono- or polybasic acid anddepending on which salt is desired—in an equimolar quantitative ratio orone differing therefrom.

As examples of salts with bases are mentioned the lithium, sodium,potassium, calcium, aluminium, magnesium, titanium, ammonium, meglumineor guanidinium salts, here, too, the bases being employed in saltpreparation in an equimolar quantitative ratio or one differingtherefrom.

It is understood that the active compounds and their pharmaceuticallyacceptable salts mentioned can also be present, for example, in the formof their pharmaceutically acceptable solvates, in particular in the formof their hydrates.

“Diseases in which pulmonary surfactant malfunction and/orphosphodiesterase 2 (PDE2) activity is detrimental” which may bementioned are in particular disorders of varying origin. Such diseasesare characterized by a pulmonary surfactant malfunction and/or animpairment of oxygenation and/or edema formation. Diseases which may bementioned as examples are ALI (Acute Lung Injury), ARDS (AdultRespiratory Distress Syndrome), IRDS (Infant Respiratory DistressSyndrome) and Asthma bronchiale. Preferred examples are ARDS and Asthmabronchiale.

The combined use of a pulmonary surfactant and a PDE2 inhibitor or theuse of a pharmaceutical composition comprising a combination aneffective amount of a pulmonary surfactant and an effective amount of aPDE2 inhibitor suited for preventing or reducing the onset of symptomsof a disease, or treating or reducing the severity of a disease in apatient in need thereof, in which disease pulmonary surfactantmalfunction and/or phosphodiesterase 2 (PDE2) activity is detrimental,reduces pulmonary surfactant malfunction and/or ameliorates oxygenationand/or prevents pulmonary edema formation and/or reverses pulmonaryedema formation.

According to this invention, an intact and well-functioning pulmonarysurfactant system is critical for normal respiration with sufficientblood oxygenation and protection from lung infection. Pulmonarysurfactant is mainly comprised of phospholipids that reduce surfacetension, prevent atelectasis and greatly reduce the work of breathing.The other major component consists of surfactant proteins, whichoptimise the biophysical function of phospholipids and/or play animportant role in host defence by acting as collectins. In this context,the term “pulmonary surfactant malfunction” refers to any condition oralteration of the surfactant system that impairs the above-mentionedproperties. In particular, impairment is a result of the inhibition ofthe surfactant activity due to infiltrated or accumulated substances inthe lung, such as proteins, proteases, and debris, or aspirated orinhaled substance (e.g. acids, saltwater, meconium, or smoke), ordilution of infiltrated, accumulated, aspirated or inhaled substances.Also particular mention is made to the impairment as a result of theinhibition of surfactant activity due to impaired surfactant production,secretion, transport, assembly, and changes in its composition.

The phrase “reducing pulmonary surfactant malfunction” refers to anyintervention or therapy, which partially or completely reduces theabove-mentioned pulmonary surfactant malfunction and thereby partiallyor completely restores pulmonary surfactant function as seen in healthyhumans.

According to this invention, oxygenation of blood can be determined by amethod known per se and familiar to the person skilled in the art bymeasuring the partial oxygen pressure in the arterial blood (PaO₂) usinga blood gas analyser or by the method as—for example—described by Hafneret al. [D. Häfner et al.: Effects of rSP-C surfactant on oxygenation andhistology in a rat lung lavage model of acute lung injury. Am. J.Respir. Crit. Care Med. 1998, 158: 270-278]. The phrase “amelioratingoxygenation” refers to an increase in PaO₂.

According to this invention, pulmonary edema is characterized by a shiftof liquid from the pulmonary vessels to the interstitial spaces and thealveolar lumen (interstitial or alveolar edema). Based on their genesis,edema may be divided in hydrostatic and permeability edema, withhydrostatic edema having cardiogenic origin (high blood pressure) andpermeability edema occurring after alterations which lead to higherpermeability of the endothelial and/or epithelial cell layer at theairway/vessel interface in the lung. Accordingly, the phrase “preventingpulmonary edema formation and/or reversing pulmonary edema” refers toany intervention, therapy, condition, or alteration that prevents and/orreverses partially or fully the above mentioned mechanisms ofliquid-transfer from the pulmonary vessels to the interstitial spacesand the alveolar lumen and thereby prevents and/or reverses bothhydrostatic and permeability edema.

The phrase “combined use” (or “combination”) embraces the administrationof a pulmonary surfactant and a PDE2 inhibitor as part of a specifictreatment regimen intended to provide a beneficial effect from theco-action of these therapeutic agents. Administration of thesetherapeutic agents in combination typically is carried out over adefined time period (usually minutes, hours, days or weeks dependingupon the combination selected). “Combined use” generally is not intendedto encompass the administration of two of these therapeutic agents aspart of separate monotherapy regimens that incidentally and arbitrarilyresult in the combinations of the present invention.

“Combined use” or “combination” within the meaning of the presentinvention is to be understood as meaning that the individual componentsof the combination can be administered simultaneously (in the form of acombination medicament—“fixed combination”) or more or lesssimultaneously, respectively in succession (from separate packunits—“free combination”; directly in succession or else alternativelyat a relatively large time interval) in a manner which is known per seand customary. As an example, one therapeutic agent could be taken inthe morning and one later in the day. Or in another scenario, onetherapeutic agent could be taken once daily and the other twice weekly.It is understood, that if individual components are administereddirectly in succession, the delay in administering the second componentshould not be such as to lose the beneficial therapeutic effect of thecombination.

It is to be understood that present invention covers all combinations ofparticular and preferred aspects of the invention described herein.Thus, present invention clearly refers to all compounds or preparationsmentioned herein as examples of a pulmonary surfactant and to allcompounds mentioned herein as a PDE2 inhibitor and to all possibleconsequential combinations. In particular, combinations which may bementioned as preferred examples of a combination of a pulmonarysurfactant and a PDE2 inhibitor are

-   a combination of erythro-9-(2-hydroxy-3-nonyl)adenine or its    pharmaceutically acceptable salts and LUSUPULTIDE,-   a combination of    9-(6-Phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one or    its pharmaceutically acceptable salts and LUSUPULTIDE,-   a combination of    6-(3,4-Dimethoxy-benzyl)-1-[1-(1-hydroxy-ethyl)-4-phenyl-butyl]-3-methyl-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one    or its pharmaceutically acceptable salts and LUSUPULTIDE-   a combination of    N-benzyl-2-(6-fluoro-2-methyl-3-pyridin-4-ylmethylene-3H-inden-1-yl)-acetamide    or its pharmaceutically acceptable salts and LUSUPULTIDE-   a combination of    (1Z)-N-benzyl-2-(6-fluoro-2-methyl-3-(3,4,5-trimethoxybenzylidene)-3H-inden-1-yl]-acetamide    or its pharmaceutically acceptable salts and LUSUPULTIDE-   a combination of    4-[N-[4-[9-[N-Methyl-N-(3-phenylpropyl)amino]hypoxanthin-2-ylmethyl]phenyl]carbamoyl]piperidine-1-carboxylic    acid benzyl ester or its pharmaceutically acceptable salts and    LUSUPULTIDE-   a combination of    4-[N-[4-[9-(N-hexyl-N-methylamino)hypoxanthin-2-ylmethyl]phenyl]carbamoyl]piperidine-1-carboxylic    acid benzyl ester or its pharmaceutically acceptable salts and    LUSUPULTIDE-   a combination of    2-(3,4-Dimethoxybenzyl)-9-[N-methyl-N-(3-phenylpropyl)amino]hypoxanthine    or its pharmaceutically acceptable salts and LUSUPULTIDE-   a combination of    9-[N-Methyl-N-(3-phenylpropyl)amino]-2-[4-(4-methylpiperazin-1-ylsulfonyl)benzyl]hypoxanthine    or its pharmaceutically acceptable salts and LUSUPULTIDE-   a combination of    2-(3,4-Dimethoxybenzyl)-7-[1(R)-[1(R)-hydroxyethyl]-4-phenylbutyl]-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one    or its pharmaceutically acceptable salts and LUSUPULTIDE,-   a combination of    7-(1-Acetylpentyl)-5-methyl-2-(4-methylbenzyl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one    or its pharmaceutically acceptable salts and LUSUPULTIDE-   a combination of    7-(1-Acetylhexyl)-5-methyl-2-(4-methylbenzyl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one    or its pharmaceutically acceptable salts and LUSUPULTIDE, and-   a combination of    2-(3,4-Dimethoxybenzyl)-7-[1-(1-hydroxyethyl)-5-hexenyl]-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one    or its pharmaceutically acceptable salts and LUSUPULTIDE.

More or less simultaneous administration of each therapeutic agent canbe effected by, for example, intratracheal or intrabronchialadministration to the subject in need thereof either as an instillationof the dissolved, liquid therapeutic agents, or as an aerosolisedsolution or as a dry powder having a fixed ratio of each therapeuticagent.

Administration of each therapeutic agent in succession, dose in time orremote in time, can be effected by any appropriate route, including, butnot limited to, intratracheal or intrabronchial instillation, oralroutes, intravenous routes, intramuscular routes, and direct absorptionor through mucous membrane tissues. The therapeutic agents can beadministered by the same route or by different routes. For example, apulmonary surfactant may be administered by intratracheal orintrabroncheal instillation while the PDE2 inhibitor may be administeredorally, intravenously, intratracheally, intrabroncheally, sublingually,intraperitoneally, or subcutaneously. The sequence in which thetherapeutic agents are administered is not narrowly critical.

The most preferred route of administration of a pulmonary surfactant isthe intratracheal or intrabronchial route by instillation in liquid formor as aerosolised solution or as dry powder. It is also preferred thatthe pulmonary surfactant is administered in form of an aerosolisedsolution or a dry powder by inhalation. Dry powder formulations ofpulmonary surfactants are preferably prepared by the spray dryingprocess as described in WO 9726863.

In case of intratracheal or intrabronchial administration of a pulmonarysurfactant preparation, it has proven advantageous to administersuspensions or solutions of the preparations according to the inventionwhich contain 10 to 100 mg of phospholipids per ml of suspension.Preferably, the preparations according to the invention are administeredper application in such an amount that the amount of phospholipids isbetween 10 and 400 mg per kilogram of body weight. As a rule,administration is carried out 1 to 3 times daily over a period of 1 to 7days. A process is preferred in which the pulmonary surfactant solutionemployed contains 0.5 to 2.0 mg of rSP-C (FF/I) per ml of solvent.Particular mention may be made of a process in which the pulmonarysurfactant solution employed contains 0.75 to 1.5 mg of rSP-C (FF/I) perml of solvent.

It has also proven advantageous to administer commercially availablepulmonary surfactant preparations in suitable dosages in accordance withdosage regimens cited in their summaries of product characteristics. Incase of intratracheal administration of BLES® the daily dose of thephospholipids will likely be in the range of 100-150 mg/kg body weight.Preferably, the daily dose will likely be 135 mg phospholipids/kg bodyweight. In case of intratracheal administration of CALFACTANT the dailydose will likely be 3-6 mL/kg body weight of CALFACTANT which is about105-210 mg phospholipids and 1,95-3,90 mg Surfactant Protein-B (SP-B)per kg body weight. In case of intratracheal administration ofSURFACTANT-TA the daily dose will likely be 60-120 mL SURFACTANT-TA perkg body weight. In case of intratracheal administration of PORACTANTALFA the daily dose will likely be 100-200 mg/kg up to a daily maximumdose of 300-400 mg/kg which is about 70-280 mg phospholipids per kg bodyweight and 1-4 g hydrophobic proteins (Surfactant Protein-B andSurfactant Protein-C) per kg body weight. In the case of intratrachealadministration of BERACTANT the daily dose will likely be 100-200 mgphospholipids per kg body weight and 4-8 mg hydrophobic proteins(Surfactant Protein-B and Surfactant Protein-C) per kg body weight. Inthe case of intratracheal administration of COLFOSCERIL PALMITATE thedaily dose will likely be 54-162 mg phospholipids per kg body weight.

PDE2 inhibitors may be administered intraduodenally, rectally, orally,transdermally, intramuscular, subcutanously, intranasally orintravenously in doses known per se and familiar to the person skilledin the art. The most preferred route of administration of a PDE2inhibitor is the oral route. In another preferred embodiment the PDE2inhibitor is administered by intravenous infusion or injection. In afurther embodiment the PDE2 inhibitor is administered by intramuscularor subcutaneous injection. Other routes of administration are alsocontemplated, including intranasal and transdermal routes, and byinhalation and by intratracheal or intrabronchial instillation.

According to U.S. Pat. No. 5,861,396, it has proved advantageous in thecase of intravenous administration to administer PDE2 inhibitors inamounts of approximately 0.01 to 10 mg/kg, preferably approximately 0.1to 10 mg/kg of body weight, to achieve effective results. In the case oforal administration of a PDE2 inhibitor, the PDE2 inhibitor may beadministered in an amount known per se and familiar to the personskilled in the art depending on the type of indication and the patientin need.

In spite of this, if appropriate it may sometimes be necessary to departfrom the amounts mentioned, mainly depending on the body weight or thetype of administration route, on individual behavior towards themedicament, the manner of its formulation and the time or interval atwhich administration takes place. Thus in some cases it may besufficient to manage with less than the abovementioned minimum amount,while in other cases the upper limit mentioned has to be exceeded. Inthe case of the administration of relatively large amounts, it may beadvisable to divide these into several individual doses over the courseof the day.

In case of pharmaceutical compositions, which are intended for oraladministration, the therapeutic agent(s) are formulated to givemedicaments according to processes known per se and familiar to theperson skilled in the art. The therapeutic agents are employed asmedicament, preferably in combination with suitable pharmaceuticalcarrier, in the form of tablets, coated tablets, capsules, granules,emulsions, suspensions, syrups or solutions, the therapeutic agentcontent advantageously being between 0.1 and 95% by weight of totalmixture and, by the appropriate choice of the carrier, it is beingpossible to achieve a pharmaceutical administration form preciselytailored to the therapeutic agent(s) and/or to the desired onset ofaction (e.g. a sustained-release form or an enteric form).

The person skilled in the art is familiar on the basis of his/her expertknowledge which carriers or excipients are suitable for the desiredpharmaceutical formulations. In addition to solvents, gel-formingagents, tablet excipients and other active compound carriers, it ispossible to use, for example, antioxidants, dispersants, emulsifiers,antifoams, flavor corrigents, preservatives, solubilizers, colorants orpermeation promoters and complexing agents (e.g. cyclodextrins).

The therapeutic agent(s) of the present invention can be administered bya variety of methods known in the art, although for many therapeuticapplications, the preferred route of administration is a freecombination of a pulmonary surfactant and a PDE2 inhibitor whereby thepulmonary surfactant is administered as a dry powder by inhalation or byintratracheal or intrabronchial instillation of a liquid and the PDE2inhibitor is administered orally. For some therapeutic application itmay be preferable to administer the pulmonary surfactant and the PDE2inhibitor in a fixed combination, whereby the preferred route ofadministration is inhalation of a dry powder formulation or anaerosolised solution or intrabronchial instillation of a liquidformulation.

The therapeutic agent(s) are dosed in an order of magnitude customaryfor the individual dose. It is more likely possible that the individualactions of the therapeutic agents are mutually positively influenced andreinforced and thus the respective doses on the combined administrationof the therapeutic agent(s) may be reduced compared with the norm.

Utility

Combinations of present invention may be prescribed to the patient in“patient pack” containing the whole course of treatment in a singlepackage. Patient packs have an advantage over traditional prescriptions,where a pharmacist divides a patient's supply of a pharmaceutical from abulk supply, in that the patient always has access to the package insertcontained in the patient pack, normally missing in traditionalprescriptions. The inclusion of a package insert has been shown toimprove patient compliance with the physician's instructions and,therefore, lead generally to more successful treatment. It will beunderstood that the administration of a combination of present inventionby means of a single patient pack, or patient packs of each componentcompound, and containing a package insert instructing the patient to thecorrect use of the invention is a desirable additional feature of theinvention leading to an increased compliance of the patient compared tothe administration of each single component.

Another beneficial effect of present invention refers to use ofcombinations of present invention. It has surprisingly been found that aunexpected therapeutic benefit, particularly a synergistic benefit, inthe prevention or reduction of the onset of symptoms of a disease, or inthe treatment or reduction of the severity of a disease in a patient inneed thereof, in which disease pulmonary surfactant malfunction and/orphosphodiesterase 2 (PDE2) activity is detrimental, can be obtained byusing a composition of a pulmonary surfactant and a PDE2 inhibitor.

For instance, it is possible by using a combination of a pulmonarysurfactant and a PDE2 inhibitor to superiorly ameliorate oxygenation ina patient in need thereof suffering from a disease in which pulmonarysurfactant malfunction and/or phosphodiesterase 2 (PDE2) activity isdetrimental compared to the use of a pulmonary surfactant or a PDE2inhibitor alone. This synergistic effect of the combination of apulmonary surfactant and a PDE2 inhibitor has been shown by in vivostudies as outlined in Example 5 and FIG. 1.

Because of this synergistic effect of a combination of presentinvention, the amount of the pulmonary surfactant may be significantlyreduced when used in a combination with a PDE2 inhibitor, which interalia significantly reduces costs of the therapy of a patient in needthereof, as pulmonary surfactants are comparatively costly. Thefrequency of ungratefulness related to the application of a pulmonarysurfactant, for example, by instillation may also be reduced compared tothe use of a pulmonary surfactant alone.

As another beneficial effect of a combination of present invention,there is provided as a result of the improved oxygenation asignificantly improvement of patients body performance - compared to theuse of a pulmonary surfactant alone or a PDE2 inhibitor alone.

Finally, it as been found that the use of a combination of a pulmonarysurfactant and a PDE2 inhibitor significantly reduces the time patientswith ARDS or IRDS have to be ventilated, and thus, it is possible by theadministration of a combination of a pulmonary surfactant and a PDE2inhibitor to avoid side effects of ventilation, for example the risk ofa nosocomial infection or pneumonia for the patients can be loweredcompared to the use of a pulmonary surfactant alone.

DESCRIPTION OF DIAGRAMS

FIG. 1: Influence of PDE-2 inhibition and VENTICUTE administration onarterial blood oxygenation after repeated saline lung lavage in rats

Male Wistar rats were prepared according to Example 5 and lungs wereravaged 5-9 times with NaCl 0.9% (=>PaO₂˜50-100 mmHg). After 60 min NaCl0.9% (open circles), VENTICUTE 12.5 mgPL/kg (filled squares,PL=Phospholipids), PODPO(9-(6-Phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one) 100 nM(stars), or VENTICUTE 12.5 mgPL/kg in combination with PODPO 100 nM(open squares) was administered intratracheally (administration volume1.2 mL). Arterial blood oxygenation (PaO₂) was determined every 30 minup to 150 min after drug administration (t=210 min). Administration ofNaCl and PODPO alone had no influence on oxygenation, but VENTICUTE 12.5mgPL/kg improved oxygenation to about 300 mmHg. The combined use of bothdrugs, i.e. VENTICUTE 12.5 mgPL/kg and PODPO 100 nM, revealed thesignificant, synergistic effect of the combination in restoringoxygenation. Data are shown as mean±SEM. * p<0.05, *** p<0.001 versusVENTICUTE 12.5 mgPL/kg.

EXAMPLES Example 1 Fixed Combination LUSUPULTIDE+PODPO For Dry PowderInhalation

9.8 g of 1,2-dipalmitoyl-3-sn-phosphatidylcholine, 4.2 g of1-paimitoyl-2-oleoyl-3-sn-phosphatidylglyce-rolammonium, 12.3 μg ofPODPO (9-(6-Phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one),0.7 g of palmitic acid, 0.36 g of calcium chloride and 0.28 g of r-SP-C(FF/I) are dissolved in 820 ml of 2-propanol/water (90:10) andspray-dried in a Büchi B 191 laboratory spray-dryer. Spray conditions:drying gas nitrogen, inlet temperature 110° C., outlet temperature59-61° C. A fine powder is obtained which can be micronized. About 55mg/kg body weight can be administered intratracheally as a dry powderwith an appropriate dry powder inhaler device for a single application.

Example 2 Fixed Combination LUSUPULTIDE+EHNA For IntrabronchialInstillation

9.8 g of 1,2-dipalmitoyl-3-sn-phosphatidylcholine, 4.2 g of1-palmitoyl-2-oleoyl-3-sn-phosphatidylglyce-rolammonium, 0.7 g ofpalmitic acid, 0.36 g of calcium chloride and 0.28 g of r-SP-C (FF/I)are spray-dried as described in Example 1. 0.88 mg EHNA(erythro-9-(2-hydroxy-3-nonyl)adenine) is dissolved in 280 mL 0.9%sodium chloride. The 15.34 g of the surfactant composition are added tothis solution and suspended. For a single application in humans 1 ml/kgbody weight of this suspension can be instilled intrabronchially guidedby a bronchoscope.

Example 3 Free Combination of BERACTANT For IntratrachealInstillation+PODPO For Oral Administration

For a single application in humans commercially available BERACTANT isadministered intratracheally 100 mg/kg as a suspension in 0.9% sodiumchloride containing 25 mg phospholipids per mL (consisting of 11.0-15.5mg/mL disaturated phosphatidycholine, 0.5-1.75 mg/mL triglycerides,1.4-3.5 mg/mL free fatty acids, and less than 1.0 mg/mL protein). Thisapplication is combined with one or several timed oral administrationsof 1 to 20 mg PODPO[9-(6-Phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin6-one].

Example 4 Free Combination PORACTANT ALPHA For IntratrachealInstillation+DHPMDP For Oral Administration

For a single application in humans commercially available PORACTANTALPHA is administered intratracheally 100-200 mg/kg. Composition per mLof suspension: phospholipid fraction from porcine lung 80 mg/mL,equivalent to about 74 mg/mL of total phospholipids and 0.9 mg/mL of lowmolecular weight hydrophobic proteins. This application is combined withone or several timed oral administrations of 1 to 20 mg DHPMDP (6-(3,4-Dimethoxy-benzyl)-1-[1-(1-hydroxy-ethyl)-4-phenyl-butyl]-3-methyl-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one).

Example 5 Rat Lung Lavage Experiment

Male Wistar rats (220-250 g) were anaesthetized, catheterised towithdraw arterial blood, and ventilated with pure oxygen (=>PaO₂˜500-550mmHg). 30 min later lungs were lavaged 5-9 times with NaCl 0.9%(=>PaO₂˜50-100 mmHg). After 60 min NaCl 0.9% (open circles), VENTICUTE12.5 mgPL/kg (filled squares, PL=Phospholipids), PODPO(9-(6-Phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one) 100 nM(stars), or VENTICUTE 12.5 mgPL/kg in combination with PODPO 100 nM(open squares) was administered intratracheally (administration volume1.2 mL). Arterial blood oxygenation (PaO₂) was determined every 30 minup to 150 min after drug administration (t=210 min). According to FIG.1, administration of NaCl and PODPO alone had no influence onoxygenation, but VENTICUTE 12.5 mgPL/kg improved oxygenation to about300 mmHg. Combination of both drugs, VENTICUTE 12.5 mgPL/kg containingPODPO 100 nM, showed a significant, synergistic effect in restoring theoxygenation. Data are shown as mean±SEM. * p<0.05, *** p<0.001 versusVENTICUTE 12.5 mgPL/kg.

1. Combined use of a pulmonary surfactant and a PDE2 inhibitor forpreventing or reducing the onset of symptoms of a disease, or treatingor reducing the severity of a disease in a patient in need thereof, inwhich disease pulmonary surfactant malfunction and/or phosphodiesterase2 (PDE2) activity is detrimental.
 2. Use of a combination of a pulmonarysurfactant and a PDE2 inhibitor for the preparation of a medicament forpreventing or reducing the onset of symptoms of a disease, or treatingor reducing the severity of a disease in a patient in need thereof, inwhich disease pulmonary surfactant malfunction and/or phosphodiesterase2 (PDE2) activity is detrimental.
 3. Method for preventing or reducingthe onset of symptoms of a disease in which pulmonary surfactantmalfunction and/or phosphodiesterase 2 (PDE2) activity is detrimental,or treating or reducing the severity of a disease in which pulmonarysurfactant malfunction and/or phosphodiesterase 2 (PDE2) activity isdetrimental by administering to a patient in need thereof an effectiveamount of (1) a pulmonary surfactant and (2) a PDE2 inhibitor.
 4. Themethod according to claim 3, wherein an effective amount of (1) apulmonary surfactant and (2) a PDE2 inhibitor is administeredsimultaneously to a patient in need thereof.
 5. The method according toclaim 3, wherein an effective amount of (1) a pulmonary surfactant and(2) a PDE2 inhibitor are administered in succession, close in time orremote in time, in any order whatever to a patient in need thereof. 6.Use or method according to any of claims 1 to 5, wherein the pulmonarysurfactant is selected from the group consisting of PORACTANT ALFA,BERACTANT, BOVACTANT, COLFOSCERIL PALMITATE, SURFACTANT-TA, CALFACTANT,PUMACTANT, LUSUPULTIDE and SINAPULTIDE.
 7. Use or method according toclaim 6, wherein the pulmonary surfactant is LUSUPULTIDE.
 8. Use ormethod according to any of claims 1 to 5, wherein the PDE2 inhibitor isselected from the group consisting ofN-Benzyl-2-[5-fluoro-2-methyl-1(Z)-(3,4,5-trimethoxybenzylidene)inden-3-yl]acetamide,2-(3′-Aminobiphenyl-4-ylmethyl)-9-(1-methyl-4-phenylbutyl)hypoxanthine,N-Benzyl-2-[5-fluoro-2-methyl-1(Z)-(3,4,5-trimethoxybenzylidene)inden-3-yl]acetamide,2-(3′-Aminobiphenyl-4-ylmethyl)-9-(1-methyl-4-phenylbutyl)hypoxanthine,2-Benzyl-9-(1-methyl-4-phenylbutyl)hypoxanthine,2-(3,4-Dichlorobenzyl)-9-[1-(1-hydroxyethyl)-4-phenylbutyl]hypoxanthine,2-(4-Fluorobenzyl)-9-(1-methyl-4-phenylbutyl)hypoxanthine,9-(1-Methyl-4-phenylbutyl)-2-[4-(3-thienyl)benzyl]hypoxanthine,1-[4-[9-[1-(1-Hydroxyethyl)-4-phenylbutyl]hypoxanthin-2-ylmethyl]-2-methoxyphenylsulfonyl]piperidine-4-carboxylicacid,2-(Biphenyl-4-yl)-9-(1-methyl-4-phenylbutyl)-6,9-dihydro-1H-purin-6-one,2-(4-Chlorophenyl)-9-[1-(1-hydroxyethyl)heptyl]-6,9-dihydro-1H-purin-6-one,2-Cyclohexyl-9-[1-(1-hydroxyethyl)-4-phenylbutyl]-6,9-dihydro-1H-purin-6-one,2-Cyclopropyl-9-(1-methyl-4-phenylbutyl)-6,9-dihydro-1H-purin-6-one,2-(1,3-Benzodioxol-5-yl)-9-(1-methyl-4-phenylbutyl)-6,9-dihydro-1H-purin-6-one,erythro-9-(2-hydroxy-3-nonyl)adenine,9-(6-Phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one,6-(3,4-Dimethoxy-benzyl)-1-[1-(1-hydroxy-ethyl)-4-phenyl-butyl]-3-methyl-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one,N-benzyl-2-(6-fluoro-2-methyl-3-pyridin-4-ylmethylene-3H-inden-1-yl)-acetamide,(1Z)-N-benzyl-2-[6-fluoro-2-methyl-3-(3,4,5-trimethoxybenzylidene)-3H-inden-1-yl]-acetamide,N-Benzyl-2-[5-fluoro-2-methyl-1-[(Z)-(pyridin-4-yl)methylene]-1H-inden-3-yl]acetamidehydrochloride,4-[N-[4-[9-[N-Methyl-N-(3-phenylpropyl)amino]hypoxanthin-2-ylmethyl]phenyl]carbamoyl]piperidine-1-carboxylicacid benzyl ester,4-[N-[4-[9-N-hexyl-N-methylamino)hypoxanthin-2-ylmethyl]phenyl]carbamoyl]piperidine-1-carboxylicacid benzyl ester,2-(3,4-Dimethoxybenzyl)-9-[N-methyl-N-(3-phenylpropyl)amino]hypoxanthine,9-[N-Methyl-N-(3-phenylpropyl)amino]-2-[4-(4-methylpiperazin-1-ylsulfonyl)benzyl]hypoxanthine,2-(3,4-Dimethoxybenzyl)-7-[1(R)-[1(R)-hydroxyethyl]4-phenylbutyl]-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one,7-(1-Acetylpentyl)-5-methyl-2-(4-methylbenzyl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one,7-(1-Acetylhexyl)-5-methyl-2-(4-methylbenzyl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one,2-(3,4-Dimethoxybenzyl)-7-[1-(1-hydroxyethyl)-5-hexenyl]-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one,and the pharmaceutically acceptable salts of these compounds.
 9. Use ormethod according to any of claims 1 to 8, wherein the PDE2 inhibitor isselected from the group consisting ofN-Benzyl-2-[5-fluoro-2-methyl-1(Z)-(3,4,5-trimethoxybenzylidene)inden-3-yl]acetamide,2-(3′-Aminobiphenyl-4-ylmethyl)-9-(1-methyl-4-phenylbutyl)hypoxanthine,N-Benzyl-2-[5-fluoro-2-methyl-1(Z)-(3,4,5-trimethoxybenzylidene)inden-3-yl]acetamide,2-(3′-Aminobiphenyl-4-ylmethyl)-9-(1-methyl-4-phenylbutyl)hypoxanthine,2-Benzyl-9-(1-methyl-4-phenylbutyl)hypoxanthine,2-(3,4-Dichlorobenzyl)-9-[1-(1-hydroxyethyl)-4-phenylbutyl]hypoxanthine,2-(4-Fluorobenzyl)-9-(1-methyl-4-phenylbutyl)hypoxanthine,9-(1-Methyl-4-phenylbutyl)-2-[4-(3-thienyl)benzyl]hypoxanthine,1-[5-[9-[1-(1-Hydroxyethyl)-4-phenylbutyl]hypoxanthin-2-ylmethyl]-2-methoxyphenylsulfonyl]piperidine-4-carboxylicacid,2-(Biphenyl-4-yl)-9-(1-methyl-4-phenylbutyl)-6,9-dihydro-1H-purin-6-one,2-(4-Chlorophenyl)-9-[1-(1-hydroxyethyl)heptyl]-6,9-dihydro-1H-purin-6-one,2-Cyclohexyl-9-[1-(1-hydroxyethyl)-4-phenylbutyl]-6,9-dihydro-1H-purin-6-one,2-Cyclopropyl-9-(1-methyl-4-phenylbutyl)-6,9-dihydro-1H-purin-6-one,2-(1,3-Benzodioxol-5-yl)-9-(1-methyl-4-phenylbutyl)-6,9-dihydro-1H-purin-6-one,erythro-9-(2-hydroxy-3-nonyl)adenine,9-(6-Phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one,6-(3,4-Dimethoxy-benzyl)-1-[1-(1-hydroxy-ethyl)-4-phenyl-butyl]-3-methyl-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one,N-benzyl-2-(6-fluoro-2-methyl-3-pyridin-4-ylmethylene-3H-inden-1-yl)-acetamide,(1Z)-N-benzyl-2-[6-fluoro-2-methyl-3-(3,4,5-trimethoxybenzylidene)-3H-inden-1-yl]-acetamide,N-Benzyl-2-[5-fluoro-2-methyl-1-[(Z)-(pyridin-4-yl)methylene]-1H-inden-3-yl]acetamidehydrochloride,4-[N-[4-[9-[N-Methyl-N-(3-phenylpropyl)amino]hypoxanthin-2-ylmethyl]phenyl]carbamoyl]piperidine-1-carboxylicacid benzyl ester,4-[N-[4-[9-(N-hexyl-N-methylamino)hypoxanthin-2-ylmethyl]phenyl]carbamoyl]piperidine-1-carboxylicacid benzyl ester,2-(3,4-Dimethoxybenzyl)-9-[N-methyl-N-(3-phenylpropyl)amino]hypoxanthine,9-[N-Methyl-N-(3-phenylpropyl)amino]-2-[4-(4-methylpiperazin-1-ylsulfonyl)benzyl]hypoxanthine,2-(3,4-Dimethoxybenzyl)-7-[1(R)-[1(R)-hydroxyethyl]-4-phenylbutyl]-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one,7-(1-Acetylpentyl)-5-methyl-2-(4-methylbenzyl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one,7-(1-Acetylhexyl)-5-methyl-2-(4-methylbenzyl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one,2-(3,4-Dimethoxybenzyl)-7-[1-(1-hydroxyethyl)-5-hexenyl]-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one,and the pharmaceutically acceptable salts of these compounds.
 10. Use ormethod according to any of claims 1 to 7, wherein the disease in whichpulmonary surfactant malfunction and/or phosphodiesterase 2 (PDE2)activity is detrimental is ARDS or Asthma bronchiale.
 11. Use or methodaccording to any of claims 1 to 9, wherein the disease in whichpulmonary surfactant malfunction and/or phosphodiesterase 2 (PDE2)activity is detrimental is selected from the group consisting of ALI,IRDS, ARDS and Asthma bronchiale.
 12. Pharmaceutical composition suitedfor the use or method according to claims 1 to 8 comprising an effectiveamount of a pulmonary surfactant and an effective amount of a PDE2inhibitor.
 13. Pharmaceutical composition according to claim 12,comprising as a fixed combination an effective amount of a pulmonarysurfactant and an effective amount of a PDE2 inhibitor, and optionally apharmaceutically acceptable carrier.
 14. Pharmaceutical compositionaccording to claim 13, which is a fixed pharmaceutical composition forintratracheally or intrabronchially instillation.
 15. Pharmaceuticalcomposition according to claim 12, comprising as a free combination aneffective amount of a pulmonary surfactant and optionally apharmaceutically acceptable carrier and an effective amount of a PDE2inhibitor and optionally a pharmaceutically acceptable carrier. 16.Pharmaceutical composition according to any of claims 12 to 15, whereinthe pulmonary surfactant is selected from the group consisting ofPORACTANT ALFA, BERACTANT, BOVACTANT, COLFOSCERIL PALMITATE,SURFACTANT-TA, CALFACTANT, PUMACTANT, LUSUPULTIDE OR SINAPULTIDE. 17.Pharmaceutical composition according to any of claims 12 to 16, whereinthe pulmonary surfactant is LUSUPULTIDE.
 18. Pharmaceutical compositionaccording to any of claims 12 to 15, wherein the PDE2 inhibitor isselected from the group consisting ofN-Benzyl-2-[5-fluoro-2-methyl-1(Z)-(3,4,5-trimethoxybenzylidene)inden-3-yl]acetamide,2-(3′-Aminobiphenyl-4-ylmethyl)-9-(1-methyl-4-phenylbutyl)hypoxanthine,N-Benzyl-2-[5-fluoro-2-methyl-1(Z)-(3,4,5-trimethoxybenzylidene)inden-3-yl]acetamide,2-(3′-Aminobiphenyl-4-ylmethyl)-9-(1-methyl-4-phenylbutyl)hypoxanthine,2-Benzyl-9-(1-methyl-4-phenylbutyl)hypoxanthine,2-(3,4-Dichlorobenzyl)-9-[1-(1-hydroxyethyl)-4-phenylbutyl]hypoxanthine,2-(4-Fluorobenzyl)-9-(1-methyl-4-phenylbutyl)hypoxanthine,9-(1-Methyl-4-phenylbutyl)-2-[4-(3-thienyl)benzyl]hypoxanthine,1-[5-[9-[1-(1-Hydroxyethyl)-4-phenylbutyl]hypoxanthin-2-ylmethyl]-2-methoxyphenylsulfonyl]piperidine-4-carboxylicacid,2-(Biphenyl-4-yl)-9-(1-methyl-4-phenylbutyl)-6,9-dihydro-1H-purin-6-one,2-(4-Chlorophenyl)-9-[1-(1-hydroxyethyl)heptyl]-6,9-dihydro-1H-purin-6-one,2-Cyclohexyl-9-[1-(1-hydroxyethyl)4-phenylbutyl]-6,9-dihydro-1H-purin-6-one,2-Cyclopropyl-9-(1-methyl-4-phenylbutyl)-6,9-dihydro-1H-purin-6-one,2-(1,3-Benzodioxol-5-yl)-9-(1-methyl-4-phenylbutyl)-6,9-dihydro-1H-purin-6-one,erythro-9-(2-hydroxy-3-nonyl)adenine,9-(6-Phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one,6-(3,4-Dimethoxy-benzyl)-1-[1-(1-hydroxy-ethyl)-4-phenyl-butyl]-3-methyl-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one,N-benzyl-2-(6-fluoro-2-methyl-3-pyridin-4-ylmethylene-3H-inden-1-yl)-acetamide,(1Z)-N-benzyl-2-[6-fluoro-2-methyl-3-(3,4,5-trimethoxybenzylidene)-3H-inden-1-yl]-acetamide,N-Benzyl-2-[5-fluoro-2-methyl-1-[(Z)-(pyridin-4-yl)methylene]-1H-inden-3-yl]acetamidehydrochloride,4-[N-[4-[9-[N-Methyl-N-(3-phenylpropyl)amino]hypoxanthin-2-ylmethyl]phenyl]carbamoyl]piperidine-1-carboxylicacid benzyl ester,4-[N-[4-[9-(N-hexyl-N-methylamino)hypoxanthin-2-ylmethyl]phenyl]carbamoyl]piperidine-1-carboxylicacid benzyl ester,2-(3,4-Dimethoxybenzyl)-9-[N-methyl-N-(3-phenylpropyl)amino]hypoxanthine,9-[N-Methyl-N-(3-phenylpropyl)amino]-2-[4-(4-methylpiperazin-1-ylsulfonyl)benzyl]hypoxanthine,2-(3,4-Dimethoxybenzyl)-7-[1(R)-[1(R)-hydroxyethyl]-4-phenylbutyl]-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one,7-(1-Acetylpentyl)-5-methyl-2-(4-methylbenzyl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one,7-(1-Acetylhexyl)-5-methyl-2-(4-methylbenzyl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one,2-(3,4-Dimethoxybenzyl)-7-[1-(1-hydroxyethyl)-5-hexenyl]-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one,and the pharmaceutically acceptable salts of these compounds. 19.Pharmaceutical composition according to any of claims 12 to 18, whereinthe PDE2 inhibitor is selected from the group consisting ofN-Benzyl-2-[5-fluoro-2-methyl-1(Z)-(3,4,5-trimethoxybenzylidene)inden-3-yl]acetamide,2-(3′-Aminobiphenyl-4-ylmethyl)-9-(1-methyl-4-phenylbutyl)hypoxanthine,N-Benzyl-2-[5-fluoro-2-methyl-1(Z)-(3,4,5-trimethoxybenzylidene)inden-3-yl]acetamide,2-(3′-Aminobiphenyl-4-ylmethyl)-9-(1-methyl-4-phenylbutyl)hypoxanthine,2-Benzyl-9-(1-methyl-4-phenylbutyl)hypoxanthine,2-(3,4-Dichlorobenzyl)-9-[1-(1-hydroxyethyl)-4-phenylbutyl]hypoxanthine,2-(4-Fluorobenzyl)-9-(1-methyl-4-phenylbutyl)hypoxanthine,9-(1-Methyl-4-phenylbutyl)-2-[4-(3-thienyl)benzyl]hypoxanthine,1-[5-[9-[1-(1-Hydroxyethyl)-4-phenylbutyl]hypoxanthin-2-ylmethyl]-2-methoxyphenylsulfonyl]piperidine-4-carboxylicacid,2-(Biphenyl-4-yl)-9-(1-methyl-4-phenylbutyl)-6,9-dihydro-1H-purin-6-one,2-(4-Chlorophenyl)-9-[1-(1-hydroxyethyl)heptyl]-6,9-dihydro-1H-purin-6-one,2-Cyclohexyl-9-[1-(1-hydroxyethyl)-4-phenylbutyl]-6,9-dihydro-1H-purin-6-one,2-Cyclopropyl-9-(1-methyl-4-phenylbutyl)-6,9-dihydro-1H-purin-6-one,2-(1,3-Benzodioxol-5-yl)-9-(1-methyl-4-phenylbutyl)-6,9-dihydro-1H-purin-6-one,erythro-9-(2-hydroxy-3-nonyl)adenine,9-(6-Phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one,6-(3,4-Dimethoxy-benzyl)-1-[1-(1-hydroxy-ethyl)-4-phenyl-butyl]-3-methyl-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one,N-benzyl-2-(6-fluoro-2-methyl-3-pyridin-4-ylmethylene-3H-inden-1-yl)-acetamide,(1Z)-N-benzyl-2-[6-fluoro-2-methyl-3-(3,4,5-trimethoxybenzylidene)-3H-inden-1-yl]-acetamide,N-Benzyl-2-[5-fluoro-2-methyl-1-[(Z)-(pyridin-4-yl)methylene]-1H-inden-3-yl]acetamidehydrochloride,4-[N-[4-[9-[N-Methyl-N-(3-phenylpropyl)amino]hypoxanthin-2-ylmethyl]phenyl]carbamoyl]piperidine-1-carboxylicacid benzyl ester,4-[N-[4-[9-(N-hexyl-N-methylamino)hypoxanthin-2-ylmethyl]phenyl]carbamoyl]piperidine-1-carboxylicacid benzyl ester,2-(3,4-Dimethoxybenzyl)-9-[N-methyl-N-(3-phenylpropyl)amino]hypoxanthine,9-[N-Methyl-N-(3-phenylpropyl)amino]-2-[4-(4-methylpiperazin-1-ylsulfonyl)benzyl]hypoxanthine,2-(3,4-Dimethoxybenzyl)-7-[1(R)-[1(R)-hydroxyethyl]-4-phenylbutyl]-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one,7-(1-Acetylpentyl)-5-methyl-2-(4-methylbenzyl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one,7-(1-Acetylhexyl)-5-methyl-2-(4-methylbenzyl)imidazo[5,1-f][1,2,4]triazin-4(3H)-one,2-(3,4-Dimethoxybenzyl)-7-[1-(1-hydroxyethyl)-5-hexenyl]-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one,and the pharmaceutically acceptable salts of these compounds.
 20. Use ofa pharmaceutical composition according to one of claims 12 to 19 for thetreatment of a disease selected from the group consisting of ALI, IRDS,ARDS and Asthma bronchiale.
 21. Method for preparing a pharmaceuticalcomposition of the claims 12 to 14 comprising the step: mixing aneffective amount of a pulmonary surfactant and a PDE2 inhibitor with apharmaceutically acceptable carrier.